We plan to carry out a detailed study of the energetics of subunit interactions in hemoglobin S tetramers and with structurally-related abnormal hemoglobins. The pattern of these subunit interactions are a sensitive reflection of the altered tertiary conformation resulting from primary sequence variation. Studies will be carried out by measuring (1) the tetramer-dimer dissociation constants as a function of oxygenation state, and (2) the oxygenation curves as a function of hemoglobin concentration. These data will provide for each hemoglobin a linkage map defining the variation of subunit interactions at each successive stage of oxygenation. We plan to study this problem with tetramers of hemoglobin S and with selected abnormal hemoglobins under a variety of conditions and in the presence of anti-sickling agents. We also will investigate the properties of isolated alpha and beta chains of these hemoglobins and will study their complete self-assembly into tetramers by microcalorimetry. Results will be compared with those of normal hemoglobin currently under investigation in our laboratory. The major goal in these study will be to define the thermodynamic states assumed by the quaternary complexes during functional transitions, and to correlate these with structural information. Study on the early stages of polymerization of hemoglobin S will be carried out with the aim of detecting and identifying the intermediate aggregates in the earliest pre-gelation states. Sensitive molecular sieve techniques have recently been developed which should permit a more definitive assessment of the pregellation phenomena than has hitherto been possible. Both kinetic and equilibrium properties will be studied using these techniques and they will be combined with oxygenation curves measured as a function of hemoglobin S concentration in the region of the tetramer-polymer transition. These studies will permit a critical evaluation of the currently simplest models for the polymerization process, and provide an accurate determination of the energetics involved. The studies will be conducted over wide ranges of environmental conditions, and in the presence of antisickling agents.